Altered gene expression profiles in stable versus acute childhood asthma

ABSTRACT

Differences in gene expression in control and asthma patients to profile, differentiate, evaluate, etc. patients with exacerbated asthma and stable asthma.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of Grant Nos.R01A146652-01A1 and R01HL72987 awarded by the National Institutes ofHealth.

In accordance with 37 CFR §§ 1.77, 1.821-1.824, and 1.52(e), applicantsstate that the Sequence Listing for this application is submitted onlyin electronic form on a CD-ROM and herein incorporate by reference theentire Sequence Listing contained on the CD-ROM. The only material onthe CD-ROM is the Sequence Listing for the instant patent application,which was created on Dec. 20, 2005 and has 616 KB. Applicants furtherstate that the Sequence Listing information recorded and submittedherein in computer readable form is identical to the written (CD-ROM)sequence listing.

FIELD OF THE INVENTION

The invention is directed to asthma gene expression profiles.

BACKGROUND

Asthma is the most common chronic disease of childhood and has a stronggenetic component. Microarray technology has been used previously toidentify gene profiles associated with asthma but were limited to adultpatients and to RNA derived from peripheral blood mononuclear cells.Because asthma most often begins in childhood, genes identified inadults may not represent genes important for asthma development.

SUMMARY OF THE INVENTION

Microarray technology was applied to childhood asthma. Gene profiles,also referred to herein as signatures, associated with childhood stableand exacerbated asthma, also referred to herein as acute asthma, weredetermined. It was also determined whether the same genes induced duringstable asthma were expressed during asthma exacerbations, or whether adistinct set of genes were activated during an asthma exacerbation.Microarray technology in a group-averaged approach, and confirmatoryreverse transcription polymerase chain reaction (RT-PCR) at anindividual patient level, provided global gene expression profiles inrespiratory epithelial cells derived from nasal respiratory epithelialcells in normal and asthmatic children.

Children with stable asthma (asthma-S), children experiencing an asthmaexacerbation (asthma-E), and non-asthmatic children were evaluated. RNAwas prepared from nasal respiratory epithelial cells isolated from eachchild, initially analyzed as pooled samples from the three groups.Further validation was performed on individual patient samples usingmicroarrays and RT-PCR.

Distinct gene clusters were identifiable in individual and pooledasthma-S and asthma-E samples. Asthma-E samples demonstrated thestrongest and most reproducible signatures, with 314 genes of 34,886measured as Present on the chip, demonstrating induction or repressionof greater than two-fold with p<0.05 in each of four individual samples.Asthma-S-regulated genes encompassed genes that overlapped with those ofAsthma-E, but were fewer (166) and less consistent with respect to theirbehavior across the Asthma-E patient samples. Independent geneexpression signatures were reflective of cells and genes poised orcommitted to activation by an asthma attack.

The information is useful for asthma diagnosis, evaluation of status andtreatment response, and design of prophylaxis and therapy. These andother advantages will be apparent in light of the following figures,tables, and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows hierarchical clustering and relative expression of geneshighly expressed in childhood asthma.

FIG. 2 shows quantitative reverse transcription polymerase chainreaction analysis of selected genes.

FIG. 3 shows hierarchical clustering of genes highly expressed inindividual children with stable and acute asthma compared with controls.

FIG. 4 shows selected upregulated and downregulated genes compared withnormal controls.

DETAILED DESCRIPTION

This application contains at least one drawing executed in color. APetition under 37 C.F.R. § 1.84 requesting acceptance of the colordrawings is filed separately on even date herewith. Copies of thispatent or patent application publication with color drawing(s) will beprovided by the Office upon request and payment of the necessary fee.

Applicants incorporate by reference in its entirety Guajardo et al.,Altered gene expression profiles in nasal respiratory epithelium reflectstable versus acute childhood asthma, J. Allergy Clin Immunol 2005;115:243-53.

Healthy and asthmatic children attending the clinics and emergencydepartment of Cincinnati Children's Hospital Medical Center (CCHMC) wereevaluated. Asthmatic children were either stable, indicated by thesubstantial absence of wheezing, or exacerbated, indicated by wheezing.Asthma was diagnosed according to American Thoracic Society criteria.Participants were included in one of three groups: 1. Stable allergicasthma (asthma-S group, N=10) inclusion criteria: a. younger than 18years of age, b. physician-diagnosed asthma currently stable (notwheezing), and c. positive skin prick testing to any of the allergensfrom an environmental panel that included dust mite, molds, cat, dog,feathers, weeds and ragweed, tree pollens and grass allergen extracts(Hollister-Stier Laboratories, Spokane Wash.); 2. Exacerbation of asthma(asthma-E group, N=10) included children acutely wheezing with similarinclusion criteria to the asthma-S group with the exception of skin testpositivity (skin testing was not performed in this group because itcould further deteriorate the acute status of asthma); and 3. Healthychildren (control group, N=10) inclusion criteria: a. younger than 18years of age, b. healthy with no acute infections or major chronicillnesses, and c. negative results to the above-described environmentalskin test prick panel. Exclusion criteria to participate in the studyincluded a. 18 years of age or older, b. the use of nasal or systemicsteroids within the last 30 days, c. nasal malformations/tumors, and d.acute infectious disease present in the asthma-S or control groups.Children with a concurrent diagnosis of allergic rhinitis were excludedif they had used nasal steroids within 30 days. The use of inhaledsteroids was not interrupted for this study. Skin prick testing wasperformed in the asthma-S and control groups using DermaPiks (GreerLaboratories, Lenoir N.C.). Histamine (1 mg/ml) and normal saline (0.9%NaCl) were used as positive and negative controls. Reactions wereconsidered positive if there was an erythematous base with a wheal ≧3 mmin diameter.

Nasal mucosa sampling was performed using a CytoSoft Brush (MedicalPackaging Corp., Camarillo Calif.) and the sample was immediately takento the laboratory for processing. Samples from children in the groupexperiencing an asthma exacerbation (asthma-E) were taken within onehour of arrival to the emergency room and before any steroids weregiven. The cells were suspended in phosphare buffered saline (PBS) andan aliquot was stained with Diff-Quick (Dade Behring Inc, Newark Del.).Cell counting was performed in five high power fields (hpf) and therelative percentages of cell types were calculated. A total eosinophilcell count was also performed.

RNA was isolated from the nasal mucosa sample using TRIZOL according tomanufacturer instructions (TRIZOL Reagent, Invitrogen Corporation,Carlsbad Calif.). Average RNA yield was 42.6 μg. In one embodiment, twomicrograms of RNA from each subject were pooled to form a group samplecontaining 20 μg. The three samples (Control, Asthma-S, and Asthma-E)were then submitted to the Affymetrix Genechip Core Facility atCincinnati Children's Hospital Medical Center for processing andmicroarray hybridization using the HG-U133A GeneChip (Affymetrix, SantaClara Calif.) according to Affymetrix guidelines. The HG-U133A chipmicroarray had a total of 22,215 probe sets (excluding controls) thatidentified 14,285 genes of which 12,735 are known. In addition, fourindividual samples from each group (Control, Asthma-S and Asthma-E) weresubmitted to the Affymetrix Genechip Core Facility at CCHMC formicroarray hybridization using the HG-U133_plus 2 GeneChip (Affymetrix)according to Affymetrix guidelines. The HG-U133_plus 2 chip microarrayhad a total of 54,675 probe sets.

Scanned output files were analyzed using Microarray Suite 5.0 software(Affymetrix). From cell image data files, gene transcript levels wereestimated as the Signal strength using the MAS5.0 (Affymetrix). Globalscaling was performed to compare genes from chip to chip. Arrays werescaled to the same target intensity value (Tgt=1500) per gene andanalyzed independently.

Second-stage data analyses were performed using GeneSpring software(Silicon Genetics, Redwood City Calif.). Initially, data from pooledasthma-S and asthma-E groups were analyzed by looking for genes thatwere most different in expression relative to the control group sample.A total of 299 cDNAs corresponding to genes that were three-fold up- ordown-regulated in the asthma-S or asthma-E groups when compared to thecontrol group were identified. These 299 highly expressed cDNAs wereclustered according to their expression profiles along the GI A-P axisby using hierarchical clustering algorithms as implemented in theGeneSpring program. Clustering of the dataset was by several differentnormalization methods. The use of raw ratios of hybridization versusreference or the log2 of these ratios provided an assessment of genesbased on their levels of expression. These 299 highly expressed cDNAswere clustered according to their relative expression profiles usingnormalization of raw ratios or the log2 of these ratios to theexpression of the control group. The number of immune-related genes wasobtained for each cluster.

GeneChip data from individual RNA samples were examined by a statisticalapproach designed to test the hypothesis that genes whose expression wasaltered in the individual samples would parallel that observed in thepooled samples. Next-generation human HG-U133-plus2 GeneChips (humanHG-U133_plus2) were analyzed using MicroArraySuite 5 and the resultingGeneChip intensities were exported to GeneSpring 7.0 and normalized tothe median expression level among the four control samples. Genes whoseexpression varied according to diagnostic group were obtained by firstselecting genes that the Affymetrix algorithm reported to be “Present”in at least two gene chips. This returned 32,435 genes from the 54,675on the chip. Next, an approximately normal distribution of geneexpression values was generated by representing each gene's expressionas the log of its expression Signal as measured by Microarray Suite.ANOVA was applied to the conditions with a probability of less than 0.01(acute vs. stable vs. control) to obtain genes differentially expressedbetween conditions in at least three out of five chips. Each of theselists were further filtered for median expression being at leasttwo-fold different between the two conditions. The resulting gene listof 1378 genes was then subjected to cluster analysis using StandardCorrelation as implemented in GeneSpring.

Gene specific primers (designed using Beacon Designer software) werechosen to span at least one intron in the genomic sequence to enable themRNA-derived product to be distinguished from any possible contaminatinggenomic product. The sequences of primers for the target genes were asfollows: lymphotactin (NM_(—)003175) AATCAAGACCTACACCATCAC SEQ ID NO: 1(sense) and TTCCTGTCCATGCTCCTG SEQ ID NO: 2 (anti-sense); histamine 4(H4) receptor (NM_(—)21624) GGTGTGATCTCCATTCCTTTG SEQ ID NO: 3 (sense)and GCCACCATCAGAGTAACAATC SEQ ID NO: 4 (anti-sense); retinoic acidreceptor (RARα) (hCT2294851) AGGAGACTGAGATTAGC SEQ ID NO: 5 (sense) andAAGAAGAAGGCGTAGG SEQ ID NO: 6 (anti-sense); CXCL11 (NM_(—)005409)GCTACAGTTGTTCAAGGCTTCC SEQ ID NO: 7 (sense) and TTGGGATTTAGGCATCGTTGTCSEQ ID NO: 8 (anti-sense); and ubiquitin C (UBC) (M26880)ATTTGGGTCGCGGTTCTTGSEQ ID NO: 9 (sense) and TGCCTTGACATTCTCGATGGT SEQ IDNO: 10 (anti-sense). Prior to cDNA synthesis (using SuperScript U, RNaseH, Invitrogen), 1-2 ug of each RNA sample was pretreated with DNase I(Invitrogen) to eliminate any potential contaminating genomic DNA.RT-PCR analysis was conducted with the iCycler (Bio-Rad) using the “iQSYBR Green Supermix” Taq polymerase mix (Bio-Rad). The amount ofdouble-stranded DNA product, indicated by SYBR Green fluorescence, wasmeasured at the end of each extension cycle. The relative message levelsof each target gene were normalized to the UBC housekeeping gene.

Statistical differences between the relative expression levels of RARα,H4R, CXCL11, and lymphotactin genes among the different groups weredetermined by ANOVA (one-way) of the means and standard error values.This was followed by the Bonferroni procedure to allow for multiplecomparisons. A p-value of <0.05 was considered significant.

The mean age in years for the Control (n=10), Asthma-S (n=10), andAsthma-E (n=10) group subjects was 11.7 (SD±2.3), 11.4 (SD±3.4), and10.1 (SD±6.17) respectively. The gender (male:female) and race(AfricanAmerican:Caucasian) ratios were 7:3 and 8:2 for the controlgroup; 7:3 and 5:5 for the Asthma-S group, and 6:4 and 9:1 for theAsthma-E group. There was no statistical difference between the groups.The children in the asthma-S and asthma-E groups were predominantlyAfrican American males, which agreed with published data regarding theracial and gender distributions of childhood asthma in urbanenvironments.

The cellular composition of the nasal respiratory epithelial sample wasdetermined for each subject. For the control group, the average numberof cells/hpf (400×) was 265 (SD±104) with 97.7% epithelial cells, 1.84%polymorphonuclear leukocyte cells (PMN), 0.36% squamous cells, and 0.07%eosinophils. For the Asthma-S group the average number of cells/hpf was219 (SD±87) with 96.3% epithelial cells, 3.32% PMN, 0.30% squamouscells, and 0.09% eosinophils; and for the Asthma-E group the averagenumber of cells/hpf was 154 (SD±69) with 92.3% epithelial cells, 7.24%PMN, 0.18% squamous cells, and 0.25% eosinophils. Epithelial cellsrepresented greater than 92% of the total cells isolated in all groups.There were higher percentages of PMN and eosinophils in samples fromchildren experiencing an asthma exacerbation, however, they remainedminor populations (7.2% and 0.25%, respectively) compared with thepercentage of respiratory cells (92.3%) and the differences were notstatistically significant. Overall, a lower average of total cells wasrecovered from the nasal samples of children experiencing an asthmaexacerbation. While not being bound by a specific theory, this may bedue to excessive mucus. RNA was isolated from each sample with anaverage yield of 42.6 μg per sample from one nostril. Two μg of RNA werepooled from each subject in each group and the three pools weresubjected to microarray analysis (HG-U133A Affymetrix GeneChip).

In the asthma-S and asthma-E pooled groups, the expression of 253 (2.0%)of the known genes changed by at least three-fold in either group. Themean raw expression of these genes was 2076 (SD 4322) with a range of17.4 to 67068 and a median and mode of 1192 and 1903, respectively.

The microarray data are summarized in FIG. 1. Hierarchical clusteringand relative expression of genes highly expressed in childhood asthmaare shown in panel A. Colors are graded to indicate increased (red) ordecreased (blue) expression relative to reference. Relative expressionin a given cluster is shown in panel B. The y-axis represents expressionnormalized to control expression. Cluster analysis examining geneprofiles revealed eight distinct clusters of genes regulated in stableand acute childhood asthma. The genes in cluster or group A (N=33) weresimilarly upregulated in both asthma-S and asthma-E. In cluster B(N=55), genes were upregulated in asthma-S, and further induced inasthma-E. Cluster C genes (N=25) were unchanged in asthma-S, butupregulated in asthma-E. Cluster D genes (N=77) were downregulated inasthma-S, but unchanged in asthma-E. Cluster E genes (N=31) wereupregulated in asthma-S, but unchanged in asthma-E. Cluster F genes(N=4) were upregulated in asthma-S, but downregulated in asthma-E.Cluster G genes (N=35) were unchanged in asthma-S, but downregulated inasthma-E. Cluster H genes (N=39) were downregulated in both asthma-S andasthma-E.

Cluster A (N=33), representing genes similarly upregulated in bothasthma-S and asthma-E, contained 32 known genes (0.25% of the totalknown genes). Of these genes, 27.3% were immune-related and 21.2% wereinvolved in signal transduction. The genes in Cluster B that wereupregulated during asthma exacerbations (asthma-E) to a higher extentthan in asthma-S were comprised of 43.6% immune-related genes. The genesin Cluster C that were upregulated during asthma exacerbations(asthma-E), but unchanged in stable asthma, were comprised of 44%immune-related genes. Clusters D-H were each comprised of less than 6.5%immune-related genes. Genes in Cluster E that were upregulated inchildren with stable asthma, but unchanged during asthma exacerbations,included 6.5% immune related genes. The genes in this profile includedmainly signal transduction genes and cell function enzymes. Clusters D,F, G and H, which contain genes that were downregulated, consist largelyof genes involved in basic cell functions and unknown genes.

Distinct clusters of genes that were differentially regulated inchildhood asthma were identified, as shown in Tables 1-14. Distinct setsof genes were activated during stable vs. exacerbated asthma,establishing that exacerbated asthma status is distinguished based onthe occurrence of strong gene expression signatures in nasal epithelialsamples. Stable asthma status also exhibited differential signatures butwith more variability. While not bound by any theory, this may suggestclinical and or mechanistic heterogeneity among the patients.

In the exacerbated asthma pooled sample, 12 genes were upregulated atleast two-fold (Table 15) and 50 genes were upregulated at leastthree-fold (Table 16) as compared to the control pooled sample. Fourteengenes in the exacerbated asthma pooled sample were downregulated by atleast two-fold (Table 17) and 7 genes were downregulated by at leastthree-fold (Table 18), compared to the control pooled sample. In thestable asthma pooled sample, 7 genes were upregulated by at leasttwo-fold (Table 19) and 11 genes were upregulated by at least three-fold(Table 20) compared to the control pooled sample. Also, 6 genes in thestable asthma pooled sample were downregulated by at least two-fold(Table 21) and 4 genes were downregulated by at least three-fold (Table22) compared to the control pooled sample.

Reverse transcription polymerase chain reaction (RT-PCR) analysisconfirmed expression of genes identified by microarray. For themicroarray analysis, equivalent amounts of RNA were pooled fromindividuals in each group. All individual RNA samples isolated fromnasal mucosal cells from participants from the control (N=10),asthma-S(N=10), and asthma-E (N=10) groups were analyzed by RT-PCR forexpression of each gene. Four genes (CXCL11, RARα, H4R, andlymphotactin) were examined: one induced in the asthma-E groupexclusively (Cluster C), one induced in the asthma-S group exclusively(Cluster E), and two simultaneously induced in both groups (Cluster A).

Quantitative RT-PCR analysis is shown in FIG. 2, with the y-axis in eachgraph representing relative message levels, normalized to the average ofduplicate UBC message levels. RT-PCR confirmed increased expression ofthe selected genes in asthma-S and/or asthma-E. CXCL11 expression wasincreased in asthma-E to a greater extent than asthma-S. RARα wasinduced in asthma-S, but not asthma-E, and lymphotactin was inducedequally in asthma-S and asthma-E. The RT-PCR data validated the genesidentified by chip array and confirmed differential expression in theasthma-E and asthma-S groups.

FIG. 3 shows hierarchical clustering of genes highly expressed inindividual children with stable and exacerbated asthma compared withcontrols, with colors graded to indicate increased (red) or decreased(blue) expression relative to reference. The clustering results from theindividual samples are presented beside the data from the RNA samplespooled from each group (N=10). As shown, the data from the individualsamples were substantially consistent both between individuals and whencompared to the pooled sample data. A stepwise filtering method was usedto derive a total of 161 genes whose expression was significantlydifferent between the groups (ANOVA, p<0.01), and in addition was ofsufficient magnitude to increase or decrease by at least two-fold in 3of the 4 individual samples.

In the exacerbated asthma individual group (versus the pooled group), 88genes were upregulated and 53 genes were downregulated compared to thecontrol group. In the stable asthma group, 21 genes were upregulated and12 genes were downregulated. More specifically, in the exacerbatedasthma group, 9 genes exhibited at least a two-fold increase (Table 1)and 79 genes showed at least a three-fold increase (Table 2) compared tocontrols. Also, 36 genes were decreased by at least two-fold (Table 3)and 15 genes were decreased by at least three-fold (Table 4) compared tocontrols. In the stable asthma group, 11 genes were upregulated by atleast two-fold (Table 5) and 10 genes were upregulated by at leastthree-fold (Table 6) compared to controls. Also, 8 genes weredownregulated by at least two-fold (Table 7) and 4 genes weredownregulated by at least three-fold (Table 8) compared to controls.Many of the changes in gene expression were specific to either stable orexacerbated asthma in that gene expression did not change in the othergroup or had the opposite change in expression. For example, 70 genesthat exhibited at least a two-fold increase in the exacerbated asthmagroup showed no change of expression in the stable asthma group (Table9). Also, 50 genes were downregulated by at least two-fold in theexacerbated asthma group but were unchanged in the stable asthma group(Table 10). For stable asthma specific genes, 4 genes were upregulatedby at least two-fold while unchanged in the exacerbated asthma group(Table 11) and 9 genes were downregulated by at least two-fold in thestable asthma group and remained unchanged in the exacerbated asthmagroup (Table 12). In some cases, the direction of change in geneexpression was the same for both the stable and exacerbated asthmagroups. For example, 16 genes showed at least a two-fold increase inexpression in both groups compared to control groups (Table 13). Fourgenes exhibited an inverse in expression levels, with a gene upregulatedby at least two-fold in the exacerbated asthma group and downregulatedby at least two-fold in the stable asthma group, or vice versa (Table14).

Among the 161 most upregulated and downregulated genes (at leastthree-fold change, p<0.01) that were consistent in at least 3 of the 4samples, classes of genes were evaluated to determine if a particularclass of genes was overrepresented. Two classes of genes were noted asshown in FIG. 4. Selected upregulated immune-related genes (A) anddownregulated cilia-related genes (B) compared with controls in at least3 out of 4 samples. Among the upregulated genes, 37 immune-related geneswere consistently upregulated at least three-fold (FIG. 4A). Among thedownregulated genes, 9 cilia-related genes were consistentlydownregulated at least three-fold (FIG. 4B).

Respiratory epithelial cells serve as an accessible alternative proxyfor lower respiratory epithelium, even if they may not fully representthe genes that are expressed in the lungs of children with asthma. Manyof the genes that were found to be induced in childhood asthma have beenimplicated in the pathogenesis of asthma in other studies, includingarginase, SOCS-3, complement 3a receptor, and lymphotactin. For the chiparray analysis, both pooled samples derived from equivalent amounts ofRNA from each individual in each group, as well as individual samples,were utilized. The gene expression signatures obtained from theindividual samples agreed with the pooled samples. RT-PCR of theindividual RNA samples further validated findings and RT-PCR confirmedthe chip array data.

The percentage of immune related genes was examined in each cluster. Inthe pooled samples, clusters that included genes induced specifically ineither asthma exacerbations (Cluster C) or genes that were induced at ahigher level during asthma exacerbations (Cluster D) contained thehighest percentages and absolute numbers of immune-related genes. Thiswas confirmed by further chip array analyses of the individual samples.The immune-related genes were overrepresented among the most upregulatedgenes. Genes that are not classified as immune genes may have direct orindirect effects on the immune system. Asthma-E samples demonstrated thestrongest and most reproducible signatures and these signatures weredistinct from Asthma-S. Among the most downregulated genes,cilia-related genes were overrepresented. Because the respiratoryepithelium is often damaged in asthma and there is an overproduction ofmucus, one might predict that genes important in ciliary function wouldbe induced. While not being bound by any theory, downregulation incilia-related genes may contribute to asthma pathogenesis by impairingmucus clearance. Alternatively, downregulation of cilia genes may be aresponse to damage and may be used for repair or remodeling.

Each cluster identifies novel potential target candidate genes forchildhood asthma. In Cluster A, the H4 receptor gene was induced nearlyten-fold. This gene was recently cloned and found to be expressed inleukocytes, including eosinophils, as well as the lung and it isinvolved in childhood asthma. In contrast, the H1, H2, and H3 receptorswere not induced. Also in Cluster A, SOCS-3 was induced nearlynine-fold. SOCS-3 expression correlates strongly with the pathology ofasthma and atopic dermatitis, as well as serum IgE levels in allergichuman patients. The complement 3α receptor 1 gene SEQ ID NO: 65 in Table2 was induced. In a previous study examining the role of C3α in asthma,C3α levels were increased following segmental airway challenge insensitized adults with asthma, and the receptor is also regulated duringthe effector phase of asthma. Several IFN-induced proteins were alsoinduced in this cluster of genes induced in asthma-E to a greater levelthan asthma-S. Because asthma exacerbations in children can beassociated with upper respiratory viral infections, some of these mayrepresent an IFN-mediated anti-viral response. Genes that were inducedexclusively during asthma exacerbations (Table 9) included integrin α4as well as several chemokines and chemokine receptors. Integrin α4(CD49d), which is important for eosinophil survival and recruitment, wasinduced 8.6 fold in children experiencing asthma exacerbation, but notin children with stable asthma. In contrast, genes that were induced inasthma-S, but not asthma-E (Table 11) did not include chemokinereceptors nor chemokines. The most strongly induced gene in this clusterwas the gene encoding RARα, which was induced approximately 28-foldcompared to non-asthmatic children. Retinoids exert multiple effectsupon lung differentiation and growth, and this receptor may contributeto lung repair or remodeling in children with ongoing stable asthma.Another gene in this cluster is arginase, supporting its roles as amediator of childhood asthma. In a recent study, adults with stableasthma had increased arginase expression in their lungs and BALFcompared with normal controls. In a previous study utilizing microarrayanalysis to identify genes important in asthma RNA isolated fromperipheral blood mononuclear cells from adults with atopic asthma,allergic rhinitis but not asthma, and healthy controls was analyzed.Decreased levels of interferon α/β receptor (ratio 0.42) were found,similar to results in Cluster H.

Although environmental factors likely contribute to the differentprevalence rates, genes with large allele frequency differences betweena Caucasian population and a Han Chinese population may be partlyresponsible for the current variation in asthma susceptibility.One-hundred and sixty-one known genes identified by microarray data wereexamined for large allele frequency differences between Caucasian andHan Chinese populations. Allele frequencies of the single nucleotidepolymorphisms (SNPs) within each gene in Caucasians and in Han Chinesewere retrieved from the public HapMap database (http://www.hapmap.org).F_(ST) was calculated as F_(ST)=σ²/pq, where σ² is the variance inallele frequency, and p and q are the average allele frequency of eachallele among subpopulations, respectively. When comparing twosubpopulations with two alleles in each, the variance in allelefrequency equals to a 2=(p₁−p₂)²/4, where p₁ is the allele frequency inthe first subpopulation, and p₂ is the frequency of the same allele inthe second subpopulation. Therefore, the F_(ST) was calculated byF_(ST)=(p₁−p₂)²/(4p(1−p)). The sample size obtained from HapMap projectwas large, i.e., 60 and 45 unrelated Caucasians and Han Chinese weregenotyped, respectively, and allowed for calculation of F_(ST) withoutadditional corrections. F_(ST) has a theoretical minimum of 0,indicating no genetic divergence, and a theoretical maximum of 1,indicating fixation for alternative alleles in different subpopulations.The observed F_(ST) is usually much less than 1 in human subpopulations.The following qualitative guidelines were used for the interpretation ofF_(ST): 0<F_(ST)<0.05: little genetic differentiation; 0.05<F_(ST)<0.15:moderate genetic differentiation; 0.15<F_(ST)<0.25: great geneticdifferentiation; and F_(ST)>0.25: very great genetic differentiation.

The F_(ST) for each identified SNPs (from HapMap database) wascalculated in each of the 161 most regulated genes related to childhoodasthma based on the gene expression profile comparisons. Among theasthma signature genes from the chip array results, there were 43 genes(39%) with large allele frequency differences (F_(ST)>0.15) betweenCaucasian and Han Chinese populations. Of these 43 genes, 17 had aF_(ST)>0.25 (Table 23) and 26 had a 0.15>F_(ST)>0.25 (Table 24). Thisproportion is higher than the average genetic differentiation betweenthese two subpopulations.

Among these 43 genes, six genes (PDE4B SEQ. ID NO. 27; SPRR2B SEQ. IDNO. 109; ADCY2 SEQ. ID NO. 46; KIF3A SEQ. ID NO. 80; DNAH5 SEQ ID NO.115; and PLAUSEQ. ID NO. 98) are located in chromosomal regions thathave been linked to asthma phenotypes and atopy phenotypes and have beenshown to either be regulated during allergic inflammation and/or toregulate release of Th2 cytokines including IL-13. These six genes havebeen shown to either be regulated during allergic inflammation and/or toregulate release of Th2 cytokines including IL-13. The followingsummarizes of each gene and its relationship to allergic inflammationand IL-13.

Phosphodiesterase 4B (PDE4B): The cyclic nucleotides, cAMP and cGMP, areimportant second messengers known to control many cellular processes. Ininflammatory cells, activation of cAMP signaling has negative modulatoryeffects on numerous steps required for immune inflammatory responses,including T cell activation and proliferation, cytokine recruitment andrecruitment of leukocyte. The cyclic nucleotide signaling system iscomplex and interlinked with many other pathways. Their signals aretightly controlled by regulating the synthesis and breakdown of thesemolecules. Phosphodiesterases are the enzymes that degrade andinactivate cyclic nucleotides. The phosphodiesterase 4 (PDE4) familyconsists of four genes (PDE4A-D) and each gene encodes multiple variantsgenerated from alternate splicing and different transcriptionalpromoters. The phenotypes of the different PDE4 null mice support uniquefunctions for each PDE4 gene. PDE4B null mice are generally healthy, butperipheral blood leukocytes derived from PDE4B null mice produce verylittle TNFα in response to LPS. Extensive studies using specific PDE4Binhibitors both in vitro and in vivo have demonstrated a potentanti-inflammatory effect as well as regulation of airway smooth muscleby PDE4B. Given the broad inhibitory effects, pharmacologic manipulationof PDE4 is a promising approach for treating chronic inflammatoryconditions including asthma. In animal models of asthma, PDE4 inhibitorshave been shown to inhibit airway inflammation and remodeling. A keyfeature of chronic inflammatory airway diseases such as asthma is mucushypersecretion. MUC5AC is the predominant mucin gene expressed inhealthy airways and is increased in asthmatic patients. Selective PDE4inhibition was shown to be effective in decreasing EGF-induced MUC5ACexpression in human airway epithelial cells. In a placebo-controlled,randomized clinical trial, PDE4 inhibition was found to be efficaciousin exercise-induced asthma; the mean percentage fall of FEV1 afterexercise was reduced by 41% as compared to placebo.

One mechanism by which PDE4 inhibitors exert an anti-inflammatory effectis by inhibiting IL-13 production in allergic diseases by T cells andbasophils. In one study, phytohaemagglutinin (PHA)-induced IL-13 releasefrom peripheral blood mononuclear cells from atopic asthma patients wasinhibited by rolipram, a PDE4 inhibitor. In another study, rolipraminhibited IL-13 production from PHA- or anti-CD3 plusanti-CD28-stimulated human T cells. Similarly, PDE4 inhibition blockedDermatophagoides pteronyssinus-induced interleukin-13 secretion inatopic dermatitis T cells.

Small proline-rich protein 2B (SPRR2B): SPRR genes encode a class ofsmall proline rich proteins that are strongly induced duringdifferentiation of human epidermal keratinocytes in vitro and in vivo.They are encoded by closely related members of a gene family closelylinked within a 300-kb DNA segment on human chromosome 1q21-q22 in aregion that has been linked to atopy phenotypes. These genes areexpressed predominantly in squamous epithelium, where they contribute tothe formation of the insoluble cornified crosslinked envelope thatprovides structural integrity and limits permeability throughtransglutaminase-induced N-glutamyl)lysine isopeptide crosslinks andinterchain disulfide bonds. Studies using primary human and murine cellsgrown at the air-liquid interface demonstrated that IL-13 directlyinduces SPRR2B. In fact SPRR2B was one of only four genes that wasinduced more strongly by IL-13 than IL-4. The fact that SPRR2B wasstrongly induced by IL-13 supported that it may be important in thepathogenesis of allergic disease. This role for SPRR2b substantiated ina recent study by Drs. Rothenberg and Wills-Karp where SPRR2B was shownto be induced in lungs of mice in a mouse model of asthma in aStat6-dependent fashion. Thus, SPRR2 is an allergen- and IL-13-inducedgene in experimental allergic responses that may be involved in diseasepathophysiology.

Adenylate Cyclase 2 (ADCY2): This gene encodes a member of the family ofadenylate cyclases, which are membrane-associated enzymes that catalyzethe formation of the secondary messenger cyclic adenosine monophosphate(cAMP). This enzyme is insensitive to Ca(2+)/calmodulin, and isstimulated by the G protein beta and gamma subunit complex. It islocated on chromosome 5p15 in a region that has been linked to atopyphenotypes. Cyclic AMP has a broad range of anti-inflammatory effects ona variety of effector cells involved in asthma. Pharmacologic inhibitionof adenylate cyclases inhibited IL-13 production from PHA- or anti-CD3plus anti-CD28-stimulated human T cells.

Kinesin family member 3A (KIF3A): KIF3 is a heterotrimeric member of thekinesin superfamily of microtubule associated motors. This functionallydiverse family of proteins mediates transport between the endoplasmicreticulum and the Golgi, transports protein complexes within cilia andflagella, and is involved in anterograde transport of membrane boundorganelles in neurons and melanosomes. Embryos lacking KIF3A die at 10days postcoitum, exhibit randomized establishment of L-R asymmetry, anddisplay numerous structural abnormalities. Interestingly, the KIF3A geneis located on 5q31 in a region that has demonstrated linkage to asthmaand atopy in multiple studies. It is located immediately upstream of IL4and IL13 and conserved non-coding sequences in this area have beenimplicated in the coordinate transcriptional regulation of IL-4 andIL-13.

Dynein, axonemal, heavy polypeptide 5 (DNAH5): DNAH5 is a component ofthe outer dynein arm of cilia. Mutations in DNAH5 resulting innon-functional proteins have been found to be responsible for primaryciliary dyskinesia. Similar to ADCY2, the DNAH5 gene is located onchromosome 5p15 in a region that has been linked to atopy phenotypes.IL-13 has direct effects on ciliary function, specifically it altersmucociliary differentiation and decreases ciliary beat frequency ofciliated epithelial cells.

Plasminogen activator, urokinase (PLAU): PLAU is located on chromosome10q24 and its gene product, urokinase, is serine protease involved indegradation of the extracellular matrix. Urokinase converts plasminogento plasmin by specific cleavage of an Arg-Val bond in plasminogen.Recent studies suggest that the plasmin system plays an active role intissue remodeling by influencing the production of inflammatorymediators and growth factors. Plasmin also degrades the extracellularmatrix (ECM), either directly removing glycoproteins from ECM or byactivating matrix metalloproteinases (MMPs). Urokinase is synthesized byairway cells, and inflammatory mediators affect its expression. In mousemodels of asthma urokinase is induced in the lungs of mice, and inmonocytes urokinase is induced in response to IL-4 and IL-13. The roleof urokinase in inflammation was further defined in a recent studywhereby urokinase gene-targeted mice fail to generate a Th2 immuneresponse following schistosomal antigen challenge. The plasmin system isalso involved in eotaxin-mediated chemotaxis of eosinophils.

Each of the gene products is expressed in respiratory epithelium. Noneof these epithelial genes have been studied as potential candidate genesfor asthma.

In summary, the distinct gene expression profiles in nasal respiratoryepithelial cells of children with stable asthma (asthma-S) and childrenexperiencing an asthma exacerbation (asthma-E) provided an overview ofthe genetic portrait of childhood asthma and the differences in genesimportant in promoting the development of asthma versus promoting theongoing phenotype of asthma. Strong gene expression signatures thatreflect clinical asthma attack status in readily sampled patient tissuesprovide a new opportunity for molecular sub-classification and clinicalmanagement of asthma patients. Both stabilized and acutely affectedasthmatic children exhibited characteristic expression profiles thataffect understanding of disease status, treatment response, and newtherapies. TABLE 1 Individual samples Exacerbated Asthma two-foldincrease Seq. ID Common Description Genbank Number EMR2 egf-like modulecontaining, mucin-like, hormone NM_013447 22 receptor-like 2 THBDthrombomodulin NM_000361 23 ZNF407 zinc finger protein 407 NM_017757 31ALDH1A3 aldehyde dehydrogenase 1 family, member A3 NM_000693 39 OLR1oxidized low density lipoprotein (lectin-like) receptor 1 NM_002543 69SCEL sciellin NM_144777 77 PLAU plasminogen activator, urokinaseNM_002658 98 DCP2 decapping enzyme hDcp2 NM_152624 126 SLC30A7 solutecarrier family 30 (zinc transporter), member 7 NM_133496 152

TABLE 2 Individual samples Exacerbated Asthma three-fold increase Seq.ID Common Description Genbank Number KRT24 keratin 24 NM_019016 14SOSTDC1 sclerostin domain containing 1 NM_015464 15 BM039uncharacterized bone marrow protein BM039 NM_018455 16 KRT6A keratin 6ANM_005554 17 CALB1 calbindin 1, 28 kDa NM_004929 18 GPR65 Gprotein-coupled receptor 65 NM_003608 20 FPRL1 formyl peptidereceptor-like 1 NM_001462 24 PROK2 prokineticin 2 NM_021935 26 PDE4Bphosphodiesterase 4B, cAMP-specific (phosphodiesterase NM_002600 27 E4dunce homolog, Drosophila) FOS v-fos FBJ murine osteosarcoma viraloncogene homolog NM_005252 28 TREM1 triggering receptor expressed onmyeloid cells 1 NM_018643 29 HAL histidine ammonia-lyase NM_002108 30SLC2A14 solute carrier family 2 (facilitated glucose transporter),NM_153449 32 member 14 AQP9 aquaporin 9 NM_020980 33 BCL2A1 BCL2-relatedprotein A1 NM_004049 34 CYP1A1 cytochrome P450, family 1, subfamily A,polypeptide 1 NM_000499 35 SERPINB4 serine (or cysteine) proteinaseinhibitor, clade B NM_002974 36 (ovalbumin), member 4 CLECSF6 C-type(calcium dependent, carbohydrate-recognition NM_016184 40 domain)lectin, superfamily member 6 IL1B interleukin 1, beta NM_000576 41LILRB1 leukocyte immunoglobulin-like receptor, subfamily B NM_D06669 42(with TM and ITIM domains), member 1 SLC2A3 solute carrier family 2(facilitated glucose transporter), NM_006931 43 member 3 S100A9 S100calcium binding protein A9 (calgranulin B) NM_002965 44 PLEK pleckstrinNM_002664 45 DTNA dystrobrevin, alpha NM_001390 47 ARNTL2 arylhydrocarbon receptor nuclear translocator-like 2 NM_020183 48 RAI3retinoic acid induced 3 NM_003979 49 GOS2 putative lymphocyte G0/G1switch gene NM_015714 50 RPEL1 RPEL repeat containing 1 AB051520 51CSF2RB colony stimulating factor 2 receptor, beta, low-affinityNM_000395 52 (granulocyte-macrophage) PLAUR plasminogen activator,urokinase receptor NM_002659 53 HIST1H2BH histone 1, H2bh NM_003524 54EMP1 epithelial membrane protein 1 NM_001423 55 TF transferrin NM_00106356 PRG1 proteoglycan 1, secretory granule NM_002727 57 unnamed proteinproduct; diaminopimelate decarboxylase (AA 1-327); Bacillus subtilis lysgene for X17013 58 diaminopimelate decarboxylase (EC 4.1.1.20). PLEKpleckstrin NM_002664 59 GPR43 G protein-coupled receptor 43 NM_005306 60HIST1H2BC histone 1, H2bc NM_003526 61 SPRR1A small proline-rich protein1A NM_005987 62 integrin, alpha M (complement component receptor 3,NM_000632 65 ITGAM alpha; also known as CD11b (p170), macrophage antigenalpha polypeptide) SOD2 superoxide dismutase 2, mitochondrial NM_00063667 GPR97 G protein-coupled receptor 97 NM_170776 70 SPEC1 small proteineffector 1 of Cdc42 NM_020239 72 MMP25 matrix metalloproteinase 25NM_022468 73 synonyms: HIS, HSI, ARL1, ARL-1, ALDRLn, AKR1B11, AKR1B12,MGC14103; aldose reductase-like 1; aldo-keto AKR1B10 reductase family 1,member B11 (aldose reductase-like); NM_004812 74 aldose reductase-likepeptide; aldose reductase-related protein; small intestine reductase;go_(—) synonyms: HIS, HSI, ARL1, ARL-1, ALDRLn, AKR1B11, AKR1B12,MGC14103; aldose reductase-like 1; aldo-keto AKR1B10 reductase family 1,member B11 (aldose reductase-like); NM_020299 75 aldose reductase-likepeptide; aldose reductase-related protein; small intestine reductase;go_(—) EMP1 epithelial membrane protein 1 NM_001423 76 HM74 putativechemokine receptor NM_006018 78 NR4A1 nuclear receptor subfamily 4,group A, member 1 NM_002135 79 HIST1H1C histone 1, H1c NM_005319 83 Homosapiens transcribed sequences BQ010718 84 SERPINB13 serine (or cysteine)proteinase inhibitor, clade B (ovalbumin), member 13 NM_012397 86HIST1H2BK histone 1, H2bk NM_080593 88 IL1R2 interleukin 1 receptor,type II NM_004633 90 DUSP5 dual specificity phosphatase 5 NM_004419 92TLR4 toll-like receptor 4 NM_003266 93 CCR1 chemokine (C—C motif)receptor 1 NM_001295 94 RAI3 retinoic acid induced 3 NM_003979 96 KRT13keratin 13 NM_153490 97 Homo sapiens transcribed sequences BQ277484 101SLC11A1 solute carrier family 11 (proton-coupled divalent metalNM_000578 105 ion transporters), member 1 C1QR1 complement component 1,q subcomponent, receptor 1 NM_012072 106 IL1A interleukin 1, alphaNM_000575 107 SPRR2B small proline-rich protein 2B NM_001017418 109SPRR3 small proline-rich protein 3 NM_005416 110 MAD MAX dimerizationprotein 1 NM_002357 112 CLECSF12 C-type (calcium dependent,carbohydrate-recognition NM_197953 114 domain) lectin, super-familymember 12 SPRR1B small proline-rich protein 1B (cornifin) NM_003125 117EAT2 SH2 domain-containing molecule EAT2 NM_053282 120 PRV1 polycythemiarubra vera 1 NM_020406 129 RAB35 RAB35, member RAS oncogene familyNM_006861 132 S100A2 S100 calcium binding protein A2 NM_005978 134 CD44CD44 antigen (homing function and Indian blood group NM_000610 135system) JUNB jun B proto-oncogene NM_002229 139 LILRB2 leukocyteimmunoglobulin-like receptor, subfamily B NM_005874 145 (with TM andITIM domains), member 2 C4.4A GPI-anchored metastasis-associated proteinhomolog NM_014400 146 CPA4 carboxypeptidase A4 NM_016352 153 LILRB1leukocyte immunoglobulin-like receptor, subfamily B NM_006669 160 (withTM and ITIM domains), member 1 FCGR2A Fc fragment of IgG, low affinityIIa, receptor for (CD32) NM_021642 163* Denotes a “corrected” accession number found in Genbank

TABLE 3 Individual samples Exacerbated Asthma two-fold decrease Seq. IDCommon Description Genbank Number SF1 splicing factor 1 NM_004630 11KCTD7 potassium channel tetramerisation domain containing 7 NM_153033 12DNAH7 dynein, axonemal, heavy polypeptide 7 NM_018897 63 FLJ23505 Homosapiens cDNA: FLJ23505 fis, clone LNG03017 NM_024716 68 KIF3A kinesinfamily member 3A NM_007054 80 IGFBP7 insulin-like growth factor bindingprotein 7 NM_001553 81 TSAP6 tumor suppressor pHyde; Homo sapiensdudulin 2 NM_182915 82 (TSAP6), mRNA. IPW Homo sapiens mRNA; cDNADKFZp686M12165 (from BX648788 89 clone DKFZp686M12165) PLTP phospholipidtransfer protein NM_006227 91 gb: BC029442.1/DB_XREF = gi: 20809535/TID= Hs2Affx.1.373/ CNT = 1/FEA = FLmRNA/TIER = FL/STK = 1/ NOTE =sequence(s) not in UniGene/DEF = Homo sapiens, BC029442 99 Similar toimmunity associated protein 1, clone MGC: 32707 IMAGE: 4618467, mRNA,complete cds./PROD = Similar to immu SIAH1 seven in absentia homolog 1(Drosophila) NM_003031 100 Homo sapiens transcribed sequences BX107340103 FLJ40427 hypothetical protein FLJ40427 NM_178504 108 DNAH5 dynein,axonemal, heavy polypeptide 5 NM_001369 115 DNAH9 dynein, axonemal,heavy polypeptide 9 NM_001372 116 Homo sapiens cDNA FLJ12411 fis, cloneMAMMA1002964. AK022473 118 LOC123872 similar to RIKEN cDNA 4930457P18NM_178452 121 CHST5 carbohydrate (N-acetylglucosamine 6-O)sulfotransferase 5 NM_024533 122 UI-H-BW0-aim-d-12-0-UI.s1 NCI_CGAP_Sub6Homo AW294722 123 sapiens cDNA clone IMAGE: 2729902 3′, mRNA sequence.KIAA1688 KIAA1688 protein AB051475 125 DCDC2 doublecortin domaincontaining 2 NM_016356 127 Homo sapiens transcribed sequences CD698727128 SPAG8 sperm associated antigen 8 NM_012436 130 ATM ataxiatelangiectasia mutated (includes complementation NM_000051 138 groups A,C and D) FLJ13621 hypothetical protein FLJ13621 NM_025009 140 HPXhemopexin NM_000613 141 DNAI2 dynein, axonemal, intermediate polypeptide2 NM_023036 143 Homo sapiens mRNA; cDNA DKFZp761D2417 (from cloneAL831856 148 DKFZp761D2417) 148 MGC16202 hypothetical protein MGC16202NM_032373 156 Homo sapiens LOH11CR1P gene, loss of heterozygosity,CA428747 157 11, chromosomal region 1 gene P product LOC146177 Homosapiens hypothetical protein LOC146177 NM_175059 158 (LOC146177), mRNA.MGC40053 hypothetical protein MGC40053 NM_152583 161 LOC339005 Homosapiens cDNA FLJ33935 fis, clone CTONG2017910. AK091254 162alternatively spliced; beta-1 form; possible membrane- C18orf1 spanningprotein; clone 22; Homo sapiens chromosome NM_181481 164 18 open readingframe 1 (C18orf1), mRNA. MYEF2 myelin expression factor 2 NM_016132 165DNAH3 dynein, axonemal, heavy polypeptide 3 NM_017539 167

TABLE 4 Individual samples Exacerbated Asthma three-fold decrease Seq.ID Common Description Genbank Number CLGN calmegin NM_004362 13 ADCY2adenylate cyclase 2 (brain) NM_020546 46 Homo sapiens transcribedsequence with weak similarity to protein sp: P39194 (H. sapiens)ALU7_HUMAN Alu BX361062 66 subfamily SQ sequence contamination warningentry MGC26733 hypothetical protein MGC26733 NM_144992 104 MYLK myosin,light polypeptide kinase NM_053025 111 SLC26A7 solute carrier family 26,member 7 NM_052832 119 Homo sapiens cDNA: FLJ22631 fis, clone HSI06451.AK026284 124 LOC138428 hypothetical protein LOC286207 AL833241 133 Homosapiens cDNA: FLJ22781 fis, clone KAIA1958. AK026434 136 Homo sapienscDNA FLJ12093 fis, clone HEMBB1002603. AK022155 137 Homo sapiens cDNA:FLJ23502 fis, clone LNG02862 AK027155 142 UI-H-EZ1-bbk-j-02-0-UI.s1NCI_CGAP_Ch2 Homo LOC165186 sapiens cDNA clone UI-H-EZ1-bbk-j-02-0-UI3′, mRNA NM_199280 144 sequence.; ESTs, Weakly similar to T00057hypothetical protein KIAA0423 - human (fragment) [H. sapiens]UI-E-EJ0-ahi-c-20-0-UI.r1 UI-E-EJ0 Homo sapiens cDNA BM712946 149 cloneUI-E-EJ0-ahi-c-20-0-UI 5′, mRNA sequence. FLJ14665 Start codon is notidentified.; Homo sapiens mRNA for AB058767 155 KIAA1864 protein,partial cds. SCARF2 scavenger receptor class F, member 2 NM_153334 159

TABLE 5 Individual samples Stable Asthma two-fold increase Seq. IDCommon Description Genbank Number SOSTDC1 sclerostin domain containing 1NM_015464 15 BM039 uncharacterized bone marrow protein BM039 NM_01845516 CALB1 calbindin 1, 28 kDa NM_004929 18 CYP1A1 cytochrome P450, family1, subfamily A, polypeptide 1 NM_000499 35 Homo sapiens transcribedsequences AI476722 37 EMP1 epithelial membrane protein 1 NM_001423 76SCEL sciellin NM_144777 77 UI-H-BW0-aim-d-12-0-UI.s1 NCI_CGAP_Sub6 HomoAW294722 123 sapiens cDNA clone IMAGE: 2729902 3′, mRNA sequence. DCP2decapping enzyme hDcp2 NM_152624 126 RAB35 RAB35, member RAS oncogenefamily NM_006861 132 SLC30A7 solute carrier family 30 (zinctransporter), member 7 NM_133496 152

TABLE 6 Individual samples Stable Asthma three-fold increase Seq. IDCommon Description Genbank Number FOS v-fos FBJ murine osteosarcomaviral oncogene homolog NM_005252 28 ZNF407 zinc finger protein 407NM_017757 31 ZCCHC2 zinc finger, CCHC domain containing 2 NM_017742 38ARNTL2 aryl hydrocarbon receptor nuclear translocator-like 2 NM_02018348 unnamed protein product; diaminopimelate decarboxylase (AA 1-327);Bacillus subtilis lys gene for diaminopimelate X17013 58 decarboxylase(EC 4.1.1.20). SPEC1 small protein effector 1 of Cdc42 NM_020239 72 Homosapiens transcribed sequences BQ277484 101 CBX5 chromobox homolog 5 (HP1alpha homolog, Drosophila) NM_012117 131 C4.4A GPI-anchoredmetastasis-associated protein homolog NM_014400 146 FLJ10525hypothetical protein FLJ10525 NM_018126 154

TABLE 7 Individual samples Stable Asthma two-fold decrease Seq. IDCommon Description Genbank Number BCL6 B-cell CLL/lymphoma 6 (zincfinger protein 51) NM_138931 21 TREM1 triggering receptor expressed onmyeloid cells 1 NM_018643 29 RPEL1 RPEL repeat containing 1 AB051520 51DNCI1 dynein, cytoplasmic, intermediate polypeptide 1 NM_004411 85 EPORerythropoietin receptor NM_000121 87 LILRB2 leukocyteimmunoglobulin-like receptor, subfamily B NM_005874 145 (with TM andITIM domains), member 2 HLA-DPB1 major histocompatibility complex, classII, DP beta 1 NM_002121 147 KCNC3 potassium voltage-gated channel,Shaw-related subfamily, NM_004977 166 member 3

TABLE 8 Individual samples Stable Asthma three-fold decrease Seq. IDCommon Description Genbank Number Homo sapiens mRNA; cDNA DKFZp566D053(from AW611486 71 clone DKFZp566D053) LOC253559 nectin-like protein 3NM_153184 95 ZNF483 zinc finger protein 483 NM_007169 150 ZNF483 zincfinger protein 483 NM_133464 151

TABLE 9 Individual samples Exacerbated Asthma increases, Stable Asthmano change Common Description Genbank Seq. ID Number KRT24 keratin 24NM_019016 14 KRT6A keratin 6A NM_005554 17 GPR65 G protein-coupledreceptor 65 NM_003608 20 EMR2 egf-like module containing, mucin-like,hormone receptor- NM_013447 22 like 2 THBD thrombomodulin NM_000361 23FPRL1 formyl peptide receptor-like 1 NM_001462 24 PROK2 prokineticin 2NM_021935 26 PDE4B phosphodiesterase 4B, cAMP-specific(phosphodiesterase NM_002600 27 E4 dunce homolog, Drosophila) HALhistidine ammonia-lyase NM_002108 30 SLC2A14 solute carrier family 2(facilitated glucose transporter), NM_153449 32 member 14 AQP9 aquaporin9 NM_020980 33 BCL2A1 BCL2-related protein A1 NM_004049 34 SERPINB4serine (or cysteine) proteinase inhibitor, clade B NM_002974 36(ovalbumin), member 4 ALDH1A3 aldehyde dehydrogenase 1 family, member A3NM_000693 39 CLECSF6 C-type (calcium dependent, carbohydrate-recognitionNM_016184 40 domain) lectin, superfamily member 6 IL1B interleukin 1,beta NM_000576 41 LILRB1 leukocyte immunoglobulin-like receptor,subfamily B (with NM_006669 42 TM and ITIM domains), member 1 SLC2A3solute carrier family 2 (facilitated glucose transporter), NM_006931 43member 3 S100A9 S100 calcium binding protein A9 (calgranulin B)NM_002965 44 PLEK pleckstrin NM_002664 45 DTNA dystrobrevin, alphaNM_001390 47 RAI3 retinoic acid induced 3 NM_003979 49 G0S2 putativelymphocyte G0/G1 switch gene NM_015714 50 CSF2RB colony stimulatingfactor 2 receptor, beta, low-affinity NM_000395 52(granulocyte-macrophage) PLAUR plasminogen activator, urokinase receptorNM_002659 53 HIST1H2BH histone 1, H2bh NM_003524 54 EMP1 epithelialmembrane protein 1 NM_001423 55 TF transferrin NM_001063 56 PRG1proteoglycan 1, secretory granule NM_002727 57 PLEK pleckstrin NM_00266459 GPR43 G protein-coupled receptor 43 NM_005306 60 HIST1H2BC histone 1,H2bc NM_003526 61 SPRR1A small proline-rich protein 1A NM_005987 62integrin, alpha M (complement component receptor 3, ITGAM alpha; alsoknown as CD11b (p170), macrophage antigen NM_000632 65 alphapolypeptide) SOD2 superoxide dismutase 2, mitochondrial NM_000636 67OLR1 oxidised low density lipoprotein (lectin-like) receptor 1 NM_00254369 GPR97 G protein-coupled receptor 97 NM_170776 70 MMP25 matrixmetalloproteinase 25 NM_022468 73 synonyms: HIS, HSI, ARL1, ARL-1,ALDRLn, AKR1B11, NM_004812 74 AKR1B12, MGC14103; aldose reductase-like1; aldo-keto AKR1B10 reductase family 1, member B11 (aldosereductase-like); NM_020299* aldose reductase-like peptide; aldosereductase-related protein; small intestine reductase; go_(—) synonyms:HIS, HSI, ARL1, ARL-1, ALDRLn, AKR1B11, AKR1B12, MGC14103; aldosereductase-like 1; aldo-keto AKR1B10 reductase family 1, member B11(aldose reductase-like); NM_020299 75 aldose reductase-like peptide;aldose reductase-related protein; small intestine reductase; go_(—) HM74putative chemokine receptor NM_006018 78 NR4A1 nuclear receptorsubfamily 4, group A, member 1 NM_002135 79 HIST1H1C histone 1, H1cNM_005319 83 Homo sapiens transcribed sequences BQ010718 84 SERPINB13serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member13 NM_012397 86 HIST1H2BK histone 1, H2bk NM_080593 88 IL1R2 interleukin1 receptor, type II NM_004633 90 DUSP5 dual specificity phosphatase 5NM_004419 92 TLR4 toll-like receptor 4 NM_003266 93 CCR1 chemokine (C—Cmotif) receptor 1 NM_001295 94 RAI3 retinoic acid induced 3 NM_003979 96KRT13 keratin 13 NM_153490 97 PLAU plasminogen activator, urokinaseNM_002658 98 SLC11A1 solute carrier family 11 (proton-coupled divalentmetal ion transporters), member 1 NM_000578 105 C1QR1 complementcomponent 1, q subcomponent, receptor 1 NM_012072 106 IL1A interleukin1, alpha NM_000575 107 SPRR2B small proline-rich protein 2B NM_001017418109 SPRR3 small proline-rich protein 3 NM_005416 110 MAD MAXdimerization protein 1 NM_002357 112 CLECSF12 C-type (calcium dependent,carbohydrate-recognition NM_197953 114 domain) lectin, super-familymember 12 SPRR1B small proline-rich protein 1B (cornifin) NM_003125 117EAT2 SH2 domain-containing molecule EAT2 NM_053282 120 PRV1 polycythemiarubra vera 1 NM_020406 129 S100A2 S100 calcium binding protein A2NM_005978 134 CD44 CD44 antigen (homing function and Indian blood groupNM_000610 135 system) JUNB jun B proto-oncogene NM_002229 139 LILRB2leukocyte immunoglobulin-like receptor, subfamily B (with NM_005874 145TM and ITIM domains), member 2 CPA4 carboxypeptidase A4 NM_016352 153LILRB1 leukocyte immunoglobulin-like receptor, subfamily B (withNM_006669 160 TM and ITIM domains), member 1 FCGR2A Fc fragment of IgG,low affinity IIa, receptor for (CD32) NM_021642 163*corrected accession number in Genbank

TABLE 10 Individual samples Exacerbated Asthma decreases, Stable Asthmano change Seq. ID Common Description Genbank Number SF1 splicing factor1 NM_004630 11 KCTD7 potassium channel tetramerisation domain containing7 NM_153033 12 CLGN calmegin NM_004362 13 ADCY2 adenylate cyclase 2(brain) NM_020546 46 DNAH7 dynein, axonemal, heavy polypeptide 7NM_018897 63 Homo sapiens transcribed sequence with weak similarity to66 protein sp: P39194 (H. sapiens) ALU7_HUMAN Alu subfamily BX361062 SQsequence contamination warning entry FLJ23505 Homo sapiens cDNA:FLJ23505 fis, clone LNG03017 NM_024716 68 KIF3A kinesin family member 3ANM_007054 80 IGFBP7 insulin-like growth factor binding protein 7NM_001553 81 TSAP6 tumor suppressor pHyde; Homo sapiens dudulin 2(TSAP6), NM_182915 82 mRNA. IPW Homo sapiens mRNA; cDNA DKFZp686M12165(from clone BX648788 89 DKFZp686M12165) PLTP phospholipid transferprotein NM_006227 91 gb: BC029442.1/DB_XREF = gi: 20809535/TID =Hs2Affx.1.373/ CNT = 1/FEA = FLmRNA/TIER = FL/STK = 1/ NOTE =sequence(s) not in UniGene/DEF = Homo sapiens, BC029442 99 Similar toimmunity associated protein 1, clone MGC: 32707 IMAGE: 4618467, mRNA,complete cds./PROD = Similar to immu SIAH1 seven in absentia homolog 1(Drosophila) NM_003031 100 Homo sapiens transcribed sequences BX107340103 MGC26733 hypothetical protein MGC26733 NM_144992 104 FLJ40427hypothetical protein FLJ40427 NM_178504 108 MYLK myosin, lightpolypeptide kinase NM_053025 111 DNAH5 dynein, axonemal, heavypolypeptide 5 NM_001369 115 DNAH9 dynein, axonemal, heavy polypeptide 9NM_001372 116 Homo sapiens cDNA FLJ12411 fis, clone MAMMA1002964.AK022473 118 SLC26A7 solute carrier family 26, member 7 NM_052832 119LOC123872 similar to RIKEN cDNA 4930457P18 NM_178452 121 CHST5carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 5 NM_024533 122Homo sapiens cDNA: FLJ22631 fis, clone HSI06451. AK026284 124 KIAA1688KIAA1688 protein AB051475 125 DCDC2 doublecortin domain containing 2NM_016356 127 Homo sapiens transcribed sequences CD698727 128 SPAG8sperm associated antigen 8 NM_012436 130 LOC138428 hypothetical proteinLOC286207 AL833241 133 Homo sapiens cDNA: FLJ22781 fis, clone KAIA1958.AK026434 136 Homo sapiens cDNA FLJ12093 fis, clone HEMBB1002603.AK022155 137 ATM ataxia telangiectasia mutated (includes complementationNM_000051 138 groups A, C and D) FLJ13621 hypothetical protein FLJ13621NM_025009 140 HPX hemopexin NM_000613 141 Homo sapiens cDNA: FLJ23502fis, clone LNG02862 AK027155 142 DNAI2 dynein, axonemal, intermediatepolypeptide 2 NM_023036 143 UI-H-EZ1-bbk-j-02-0-UI.s1 NCI_CGAP_Ch2 Homosapiens LOC165186 cDNA clone UI-H-EZ1-bbk-j-02-0-UI NM_199280 144 3′,mRNA sequence.; ESTs, Weakly similar to T00057 hypothetical proteinKIAA0423 - human (fragment) [H. sapiens] Homo sapiens mRNA; cDNADKFZp761D2417 (from clone DKFZp761D2417) AL831856 148UI-E-EJO-ahi-c-20-0-UI.r1 UI-E-EJO Homo sapiens cDNA BM712946 149 cloneUI-E-EJO-ahi-c-20-0-UI 5′, mRNA sequence. FLJ14665 Start codon is notidentified.; Homo sapiens mRNA for AB058767 155 KIAA1864 protein,partial cds. MGC16202 hypothetical protein MGC16202 NM_032373 156 Homosapiens LOH11CR1P gene, loss of heterozygosity, 11, CA478747 157chromosomal region 1 gene P product LOC146177 Homo sapiens hypotheticalprotein LOC146177 NM_175059 158 (LOC146177), mRNA. SCARF2 scavengerreceptor class F, member 2 NM_153334 159 MGC40053 hypothetical proteinMGC40053 NM_152583 161 LOC339005 Homo sapiens cDNA FLJ33935 fis, cloneCTONG2017910. AK091254 162 alternatively spliced; beta-1 form; possiblemembrane- C18orf1 spanning protein; clone 22; Homo sapiens chromosome 18NM_181481 164 open reading frame 1 (C18orf1), mRNA. MYEF2 myelinexpression factor 2 NM_016132 165 DNAH3 dynein, axonemal, heavypolypeptide 3 NM_017539 167

TABLE 11 Individual samples Stable Asthma increases, Exacerbated Asthmano change Seq. ID Common Description Genbank Number Homo sapienstranscribed sequences AI476722 37 ZCCHC2 zinc finger, CCHC domaincontaining 2 NM_017742 38 CBX5 chromobox homolog 5 (HP1 alpha homolog,Drosophila) NM_012117 131 FLJ10525 hypothetical protein FLJ10525NM_018126 154

TABLE 12 Individual samples Stable Asthma decreases, Exacerbated Asthmano change Seq. ID Common Description Genbank Number BCL6 B-cellCLL/lymphoma 6 (zinc finger protein 51) NM_138931 21 Homo sapiens mRNA;cDNA DKFZp566D053 (from clone AW611486 71 DKFZp566D053) DNCI1 dynein,cytoplasmic, intermediate polypeptide 1 NM_004411 85 EPOR erythropoietinreceptor NM_000121 87 LOC253559 nectin-like protein 3 NM_153184 95HLA-DPB1 major histocompatibility complex, class II, DP beta 1 NM_002121147 ZNF483 zinc finger protein 483 NM_007169 150 ZNF483 zinc fingerprotein 483 NM_133464 151 KCNC3 potassium voltage-gated channel,Shaw-related subfamily, NM_004977 166 member 3

TABLE 13 Individual samples Stable Asthma increases, Exacerbated Asthmaincreases Seq. ID Common Description Genbank Number SOSTDC1 sclerostindomain containing 1 NM_015464 15 BM039 uncharacterized bone marrowprotein BM039 NM_018455 16 CALB1 calbindin 1, 28 kDa NM_004929 18 FOSv-fos FBJ murine osteosarcoma viral oncogene homolog NM_005252 28 ZNF407zinc finger protein 407 NM_017757 31 CYP1A1 cytochrome P450, family 1,subfamily A, polypeptide 1 NM_000499 35 ARNTL2 aryl hydrocarbon receptornuclear translocator-like 2 NM_020183 48 unnamed protein product;diaminopimelate decarboxylase (AA 1-327); Bacillus subtilis lys gene fordiaminopimelate X17013 58 decarboxylase (EC 4.1.1.20). SPEC1 smallprotein effector 1 of Cdc42 NM_020239 72 EMP1 epithelial membraneprotein 1 NM_001423 76 SCEL sciellin NM_144777 77 Homo sapienstranscribed sequences BQ277484 101 DCP2 decapping enzyme hDcp2 NM_152624126 RAB35 RAB35, member RAS oncogene family NM_006861 132 C4.4AGPI-anchored metastasis-associated protein homolog NM_014400 146 SLC30A7solute carrier family 30 (zinc transporter), member 7 NM_133496 152

TABLE 14 Individual samples Inverse Changes Stable Asthma increases,Exacerbated Asthma Decrease or Stable Asthma decreases, ExacerbatedAsthma increases Seq. ID Common Description Genbank Number TREM1triggering receptor expressed on myeloid cells 1 NM_018643 29 RPEL1 RPELrepeat containing 1 AB051520 51 UI-H-BW0-aim-d-12-0-UI.s1 NCI_CGAP_Sub6Homo sapiens AW294722 123 cDNA clone IMAGE: 2729902 3′, mRNA sequence.LILRB2 leukocyte immunoglobulin-like receptor, subfamily B (with TMNM_005874 145 and ITIM domains), member 2

TABLE 15 Pooled samples Exacerbated Asthma two-fold increase Seq. IDCommon Description Genbank Number GPR65 G protein-coupled receptor 65NM_003608 20 HAL histidine ammonia-lyase NM_002108 30 SLC2A14 solutecarrier family 2 (facilitated glucose transporter), NM_153449 32 member14 PLAUR plasminogen activator, urokinase receptor NM_002659 53 TFtransferrin NM_001063 56 PLEK pleckstrin NM_002664 59 HIST1H2BC histone1, H2bc NM_003526 61 GPR97 G protein-coupled receptor 97 NM_170776 70HIST1H2BK histone 1, H2bk NM_080593 88 IL1R2 interleukin 1 receptor,type II NM_004633 90 DUSP5 dual specificity phosphatase 5 NM_004419 92TLR4 toll-like receptor 4 NM_003266 93

TABLE 16 Pooled samples Exacerbated Asthma three-fold increase Seq. IDCommon Description Genbank Number KRT24 keratin 24 NM_019016 14 SOSTDC1sclerostin domain containing 1 NM_015464 15 KRT6A keratin 6A NM_00555417 CALB1 calbindin 1, 28 kDa NM_004929 18 FCGR2B Fc fragment of IgG, lowaffinity IIb, receptor for (CD32) NM_004001 19 EMR2 egf-like modulecontaining, mucin-like, hormone receptor- NM_013447 22 like 2 FPRL1formyl peptide receptor-like 1 NM_001462 24 PDE4B phosphodiesterase 4B,cAMP-specific (phosphodiesterase E4 NM_002600 27 dunce homolog,Drosophila) FOS v-fos FBJ murine osteosarcoma viral oncogene homologNM_005252 28 TREM1 triggering receptor expressed on myeloid cells 1NM_018643 29 ZNF407 zinc finger protein 407 NM_017757 31 AQP9 aquaporin9 NM_020980 33 BCL2A1 BCL2-related protein A1 NM_004049 34 CYP1A1cytochrome P450, family 1, subfamily A, polypeptide 1 NM_000499 35ALDH1A3 aldehyde dehydrogenase 1 family, member A3 NM_000693 39 CLECSF6C-type (calcium dependent, carbohydrate-recognition NM_016184 40 domain)lectin, superfamily member 6 IL1B interleukin 1, beta NM_000576 41LILRB1 leukocyte immunoglobulin-like receptor, subfamily B (withNM_006669 42 TM and ITIM domains), member 1 SLC2A3 solute carrier family2 (facilitated glucose transporter), NM_006931 43 member 3 S100A9 S100calcium binding protein A9 (calgranulin B) NM_002965 44 PLEK pleckstrinNM_002664 45 DTNA dystrobrevin, alpha NM_001390 47 RAI3 retinoic acidinduced 3 NM_003979 49 G0S2 putative lymphocyte G0/G1 switch geneNM_015714 50 RPEL1 RPEL repeat containing 1 AB051520 51 CSF2RB colonystimulating factor 2 receptor, beta, low-affinity NM_000395 52(granulocyte-macrophage) EMP1 epithelial membrane protein 1 NM_001423 55PRG1 proteoglycan 1, secretory granule NM_002727 57 unnamed proteinproduct; diaminopimelate decarboxylase (AA 1-327); Bacillus subtilis lysgene for diaminopimelate X17013 58 decarboxylase (EC 4.1.1.20). GPR43 Gprotein-coupled receptor 43 NM_005306 60 SPRR1A small proline-richprotein 1A NM_005987 62 SOD2 superoxide dismutase 2, mitochondrialNM_000636 67 OLR1 oxidised low density lipoprotein (lectin-like)receptor 1 NM_002543 66 synonyms: HIS, HSI, ARL1, ARL-1, ALDRLn,AKR1B11, AKR1B12, MGC14103; aldose reductase-like 1; aldo-keto AKR1B10reductase family 1, member B11 (aldose reductase-like); NM_004812 74aldose reductase-like peptide; aldose reductase-related protein; smallintestine reductase; go_(—) synonyms: HIS, HSI, ARL1, ARL-1, ALDRLn,AKR1B11, AKR1B12, MGC14103; aldose reductase-like 1; aldo-keto AKR1B10reductase family 1, member B11 (aldose reductase-like); NM_020299 75aldose reductase-like peptide; aldose reductase-related protein; smallintestine reductase; go_(—) EMP1 epithelial membrane protein 1 NM_00142376 HM74 putative chemokine receptor NM_006018 78 HIST1H1C histone 1, H1cNM_005319 83 CCR1 chemokine (C—C motif) receptor 1 NM_001295 94 KRT13keratin 13 NM_153490 97 PLAU plasminogen activator, urokinase NM_00265898 IL1A interleukin 1, alpha NM_000575 107 SPRR2B small proline-richprotein 2B NM_001017418 109 SPRR1B small proline-rich protein 1B(cornifin) NM_003125 117 S100A2 S100 calcium binding protein A2NM_005978 134 LILRB2 leukocyte immunoglobulin-like receptor, subfamily B(with NM_005874 145 TM and ITIM domains), member 2 C4.4A GPI-anchoredmetastasis-associated protein homolog NM_014400 146 CPA4carboxypeptidase A4 NM_016352 153 LILRB1 leukocyte immunoglobulin-likereceptor, subfamily B (with NM_006669 160 TM and ITIM domains), member 1FCGR2A Fc fragment of IqG, low affinity IIa, receptor for (CD32)NM_021642 163* corrected accession number in Genbank

TABLE 17 Pooled samples Exacerbated Asthma two-fold decrease Seq. IDCommon Description Genbank Number CLGN calmegin NM_004362 13 FLJ13615hypothetical protein FLJ13615 NM_025114 25 FLJ23505 Homo sapiens cDNA:FLJ23505 fis, clone LNG03017 NM_024716 68 KIF3A kinesin family member 3ANM_007054 80 IGFBP7 insulin-like growth factor binding protein 7NM_001553 81 TSAP6 tumor suppressor pHyde; Homo sapiens dudulin 2(TSAP6), NM_182915 82 mRNA. PLTP phospholipid transfer protein NM_00622791 SIAH1 seven in absentia homolog 1 (Drosophila) NM_003031 100 TCF2transcription factor 2, hepatic; LF-B3; variant hepatic NM_006481 113nuclear factor LOC123872 similar to RIKEN cDNA 4930457P18 NM_178452 121DCDC2 doublecortin domain containing 2 NM_016356 127 HPX hemopexinNM_000613 141 DNAI2 dynein, axonemal, intermediate polypeptide 2NM_023036 143 alternatively spliced; beta-1 form; possible membrane-C18orf1 spanning protein; clone 22; Homo sapiens chromosome 18 NM_181481164 open reading frame 1 (C18orf1), mRNA.

TABLE 18 Pooled samples Exacerbated Asthma three-fold decrease Seq. IDCommon Description Genbank Number SF1 splicing factor 1 NM_004630 11KCTD7 potassium channel tetramerisation domain containing 7 NM_153033 12MYLK myosin, light polypeptide kinase NM_053025 111 Homo sapiens cDNAFLJ12411 fis, clone MAMMA1002964. AK022473 118

TABLE 19 Pooled samples Stable Asthma two-fold increase Seq. ID CommonDescription Genbank Number FOS v-fos FBJ murine osteosarcoma viraloncogene homolog NM_005252 28 AQP9 aquaporin 9 NM_020980 33 IL1Binterleukin 1, beta NM_000576 41 S100A9 S100 calcium binding protein A9(calgranulin B) NM_002965 44 PLEK pleckstrin NM_002664 45 OLR1 oxidisedlow density lipoprotein (lectin-like) receptor 1 NM_002543 69 JUNB jun Bproto-oncogene NM_002229 139

TABLE 20 Pooled samples Stable Asthma three-fold increase Seq. ID CommonDescription Genbank Number KRT6A keratin 6A NM_005554 17 CALB1 calbindin1, 28 kDa NM_004929 18 PDE4B phosphodiesterase 4B, cAMP-specific(phosphodiesterase E4 NM_002600 27 dunce homolog, Drosophila) HALhistidine ammonia-lyase NM_002108 30 ZNF407 zinc finger protein 407NM_017757 31 CYP1A1 cytochrome P450, family 1, subfamily A, polypeptide1 NM_000499 35 LILRB1 leukocyte immunoglobulin-like receptor, subfamilyB (with TM NM_006669 42 and ITIM domains), member 1 DTNA dystrobrevin,alpha NM_001390 47 unnamed protein product; diaminopimelatedecarboxylase (AA 1-327); Bacillus subtilis lys gene for diaminopimelateX17013 58 decarboxylase (EC 4.1.1.20). IL1A interleukin 1, alphaNM_000575 107 LILRB2 leukocyte immunoglobulin-like receptor, subfamily B(with TM NM_005874 145 and ITIM domains), member 2

TABLE 21 Pooled samples Stable Asthma two-fold decrease Seq. ID CommonDescription Genbank Number SF1 splicing factor 1 NM_004630 11 FCGR2B Fcfragment of IgG, low affinity IIb, receptor for (CD32) NM_004001 19 BCL6B-cell CLL/lymphoma 6 (zinc finger protein 51) NM_138931 21 IPW Homosapiens mRNA; cDNA DKFZp686M12165 (from clone BX648788 89DKFZp686M12165) TCF2 transcription factor 2, hepatic; LF-B3; varianthepatic nuclear NM_006481 113 factor SPAG8 sperm associated antigen 8NM_012436 130

TABLE 22 Pooled samples Stable Asthma three-fold decrease Seq. ID CommonDescription Genbank Number SOSTDC1 sclerostin domain containing 1NM_015464 15 RPEL1 RPEL repeat containing 1 AB051520 51 PLAU plasminogenactivator, urokinase NM_002658 98 C12orf6 chromosome 12 open readingframe 6 NM_020367 102

TABLE 23 Genes F_(ST) > 0.25 between Caucasians and Han Chinese from 161gene dataset Seq. ID Common Description Genbank Number PDE4Bphosphodiesterase 4B, cAMP-specific (phosphodiesterase E4 NM_002600 27dunce homolog, Drosophila) ADCY2 adenylate cyclase 2 (brain) NM_02054646 KIF3A kinesin family member 3A NM_007054 80 HIST1H1C histone 1, H1cNM_005319 83 PLAU plasminogen activator, urokinase NM_002658 98 FLJ40427hypothetical protein FLJ40427 NM_178504 108 SPRR2B small proline-richprotein 2B NM_001017418 109 MYLK myosin, light polypeptide kinaseNM_053025 111 DNAH5 dynein, axonemal, heavy polypeptide 5 NM_001369 115SLC26A7 solute carrier family 26, member 7 NM_052832 119 CHST5carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 5 NM_024533 122ZNF483 zinc finger protein 483 NM_007169 150 ZNF483 zinc finger protein483 NM_133464 151 SCARF2 scavenger receptor class F, member 2 NM_153334159 C18orf1 alternatively spliced; beta-1 form; possible membrane-NM_181481 164 spanning protein; clone 22; Homo sapiens chromosome 18open reading frame 1 (C18Sorf1), mRNA. MYEF2 myelin expression factor 2NM_016132 165 KCNC3 potassium voltage-gated channel, Shaw-relatedsubfamily, NM_004977 166 member 3* corrected accession number in Genbank

TABLE 24 Genes 0.15 < F_(ST) < 0.25 between Caucasians and Han Chinesefrom 161 gene dataset Seq. ID Common Description Genbank Number CALB1calbindin 1, 28 kDa NM_004929 18 HAL histidine ammonia-lyase NM_00210830 ZNF407 zinc finger protein 407 NM_017757 31 SLC2A14 solute carrierfamily 2 (facilitated glucose transporter), NM_153449 32 member 14BCL2A1 BCL2-related protein A1 NM_004049 34 ZCCHC2 zinc finger, CCHCdomain containing 2 NM_017742 38 ALDH1A3 aldehyde dehydrogenase 1family, member A3 NM_000693 39 PLEK pleckstrin NM_002664 45 DTNAdystrobrevin, alpha NM_001390 47 ARNTL2 aryl hydrocarbon receptornuclear translocator-like 2 NM_020183 48 EMP1 epithelial membraneprotein 1 NM_001423 55 PRG1 proteoglycan 1, secretory granule NM_00272757 DNAH7 dynein, axonemal, heavy polypeptide 7 NM_018897 63 SCELsciellin NM_144777 77 DNCI1 dynein, cytoplasmic, intermediatepolypeptide 1 NM_004411 85 MGC26733 hypothetical protein MGC26733NM_144992 104 TCF2 transcription factor 2, hepatic; LF-B3; varianthepatic NM_006481 113 nuclear factor CLECSF12 C-type (calcium dependent,carbohydrate-recognition NM_197953 114 domain) lectin, superfamilymember 12 DNAH9 dynein, axonemal, heavy polypeptide 9 NM_001372 116 EAT2SH2 domain-containing molecule EAT2 NM_053282 120 PRV1 polycythemiarubra vera 1 NM_020406 129 DNAI2 dynein, axonemal, intermediatepolypeptide 2 NM_023036 143 UI-H-EZ1-bbk-j-02-0-UI.s1 NCI_CGAP_Ch2 Homosapiens LOC165186 cDNA clone UI-H-EZ1-bbk-j-02-0-UI 3′, mRNA sequence.;NM_199280 144 ESTs, Weakly similar to T00057 hypothetical proteinKIAA0423 - human (fragment) [H. sapiens] SLC30A7 solute carrier family30 (zinc transporter), member 7 NM_133496 152 LILRB1 leukocyteimmunoglobulin-like receptor, subfamily B (with NM_006669 160 TM andITIM domains), member 1 DNAH3 dynein, axonemal, heavy polypeptide 3NM_017539 167

Other variations or embodiments of the invention will also be apparentto one of ordinary skill in the art from the above figures, tables, anddescription. Thus, the forgoing embodiments are not to be construed aslimiting the scope of this invention.

1. A method to evaluate a patient comprising: obtaining from a tissue anexpression profile of at least one gene activated in lungs of a humanpatient with asthma, comparing the patient's gene expression profilewith a gene expression profile for stable asthma and a gene expressionprofile for exacerbated asthma, and determining whether the patient hasa propensity for at least one of stable asthma or exacerbated asthmabased on at least a two-fold difference between at least one gene in theexpression profile.
 2. The method of claim 1 based on at least athree-fold difference.
 3. The method of claim 1 wherein the tissue isnasal respiratory epithelium.
 4. The method of claim 1 comparing acluster of genes.
 5. The method of claim 1 or claim 2 wherein thedifference between at least one of SEQ ID NOS: 11-18, 20, 22-24, 26-36,39-63, 65-70, 72-84, 86, 88-94, 96-101, 103-112, 114-130, 132-146,148-149, 152-153, 155-165, or 167 gene indicates a propensity forexacerbated asthma.
 6. The method of claim 1 or claim 2 wherein thedifference between at least one of SEQ ID NOS: 15-16, 18, 21, 28, 29,31, 35, 37-38, 48, 51, 58, 71-72, 76-77, 85, 87, 95, 101, 123, 126,131-132, 145-147, 150-152, 154, or 166 gene indicates a propensity forstable asthma.
 7. The method of claim 1 wherein the patient is a child.8. A diagnostic method comprising obtaining a gene expression profilefrom nasal respiratory epithelium of a patient, and diagnosingexacerbated asthma in the patient if at least a two-fold increasebetween at least one of SEQ ID NOS: 14-18, 20, 22-24, 26-36, 39-45,47-62, 65, 67, 69-70, 72-79, 83-84, 86, 88, 90, 92-94, 96-98, 101,105-107, 109-110, 112, 114, 117, 120, 126, 129, 132, 134-135, 139,145-146, 152-153, 160, or 163 in the patient tissue expression profileover a control tissue expression profile is present.
 9. A diagnosticmethod comprising obtaining a gene expression profile from nasalrespiratory epithelium of a patient, and diagnosing exacerbated asthmain the patient if at least a two-fold decrease in a at least one of SEQID NO: 11-13, 46, 63, 66, 68, 80-82, 89, 91, 99-100, 103-104, 108, 111,115-116, 118-119, 121-125, 127-128, 130, 133, 136-138, 140-144, 148-149,155-159, 161-162, 164-165, or 167 in the patient tissue expressionprofile over a control tissue expression profile is present.
 10. Adiagnostic method comprising obtaining a gene expression profile fromnasal respiratory epithelium of a patient, and diagnosing stable asthmain the patient if at least a two-fold increase between at least one ofSEQ ID NOS: 15-16, 18, 28, 31, 35, 37-38, 48, 58, 72, 76-77, 101, 123,126, 131-132, 146, 152, or 154 in the patient tissue expression profileover a control tissue expression profile is present.
 11. A diagnosticmethod comprising obtaining a gene expression profile from nasalrespiratory epithelium of a patient, and diagnosing stable asthma in thepatient if at least a two-fold decrease in at least one of SEQ ID NOS:21, 29, 51, 71, 85, 87, 95, 145, 147, 150-151, or 166 in the patienttissue expression profile over a control tissue expression profile ispresent.
 12. The method of any one of claims 8, 9, 10, or 11 furthercomprising identifying at least one gene for prophylactic or therapeuticintervention.
 13. A cluster of genes differentially regulated in atleast one of stable asthma or acute asthma comprising SEQ ID NOS:11-167.
 14. The cluster of genes of claim 10 having an allele frequencydifference >0.15 between a Caucasian population and a Han Chinesepopulation, the genes comprising SEQ ID NOS: 18, 27, 30-32, 34, 38-39,45-48, 55, 57, 63, 77, 80, 83, 85, 98, 104, 108-109, 111, 113-116,119-120, 122, 129, 143-144, 150-152, 159-160, and 164-167.
 15. Thecluster of genes of claim 10 located in a chromosomal region linked toat least one of an asthma phenotype or an atopy phenotype.
 16. Thecluster of genes of claim 13 comprising PDE4B SEQ. ID NO: 27, SPRR2BSEQ. ID NO: 109, ADCY2 SEQ. ID NO: 46, KIF3A SEQ. ID NO: 80, DNAH5 SEQID NO: 115, and PLAU SEQ. ID NO:
 98. 17. A cluster of genes induced atleast two-fold in a patient with exacerbated asthma over a patient withstable asthma, the cluster comprising SEQ ID NOS: 14, 17, 20, 22-24,26-27, 30, 32-34, 36, 39-45, 47, 49-50, 52-57, 59-62, 65, 67, 69-70,73-75, 78-79, 83-84, 86, 88, 90, 92-94, 96-98, 105-107, 109-110, 112,114, 117, 120, 129, 134-135, 139, 145, 153, 160, and
 163. 18. A clusterof genes repressed at least two-fold in a patient with exacerbatedasthma over a patient with stable asthma, the cluster comprising SEQ IDNOS: 11-13, 46, 63, 66, 68, 80-82, 89, 91, 99-100, 103-104, 108, 111,115-116, 118-119, 121-122, 124-125, 127-128, 130, 133, 136-138, 140-144,148-149, 155-159, 161-162, 164-165, and
 167. 19. A target candidate genefor preferential intervention in childhood exacerbated asthma overchildhood stable asthma comprising integrin α4 for preferentialintervention in childhood exacerbated asthma over childhood stableasthma.
 20. A cluster of genes induced at least two-fold in a patientwith stable asthma over a patient with exacerbated asthma, the clustercomprising SEQ ID NOS: 37, 38, 131, and
 154. 21. A cluster of genesrepressed at least two-fold in a patient with stable asthma over apatient with exacerbated asthma, the cluster comprising SEQ ID NOS: 21,71, 85, 87, 95, 147, 150, 151, and
 166. 22. A cluster of genes inducedat least two-fold in a patient with stable asthma over a patient withexacerbated asthma wherein the cluster lacks genes encoding chemokinereceptors or chemokines.
 23. A gene for preferential intervention inchildhood stable asthma over childhood exacerbated asthma comprising agene encoding retinoic acid receptor α for preferential intervention inchildhood stable asthma over childhood exacerbated asthma.
 24. A clusterof genes induced at least two-fold in both exacerbated asthma and stableasthma comprising SEQ ID NOS: 15-16, 18, 28, 31, 35, 48, 58, 72, 76-77,101, 126, 132, 146, and
 152. 25. A cluster of genes inversely expressedbetween patients with exacerbated asthma and patients with stableasthma, the expression being at least a two-fold difference, the clustercomprising SEQ ID NOS: 29, 51, 123, and
 145. 26. A gene for preferentialintervention in at least one of childhood exacerbated asthma orchildhood stable asthma, the gene comprising at least one of histamine 4receptor gene or SOCS-3 gene.
 27. A cluster of genes induced at leastthree-fold in a patient during asthma exacerbation and induced less thanthree-fold in the patient during stable asthma, the genes comprising SEQID NOS: 15, 16, 18, 35, 76, and
 132. 28. An evaluation method comprisingidentifying at least one gene expression profile in a nasal epithelialcell of a child patient with asthma altered over a gene expressionprofile in a nasal epithelial cell of a child control, and using theidentification to evaluate the child patient as having a propensity forat least one of stable asthma or exacerbated asthma based on at least atwo-fold difference in at least one gene in the expression profile. 29.The method of claim 28 based on at least a three-fold difference. 30.The method of claim 28 further comprising treating the patient forstable asthma or exacerbated asthma based on the evaluation.
 31. Themethod of claim 28 further comprising prophylactically treating thepatient for stable asthma or exacerbated asthma based on the evaluation.32. A method to assess lower respiratory epithelial cell DNA comprisingobtaining DNA from nasal mucosa cells and assessing the DNA, the nasalepithelial cell DNA as an accessible proxy for DNA from lowerrespiratory epithelial cells.
 33. The method of claim 32 wherein nasalmucosa cells are obtained by nasal mucosa sampling.
 34. A method toclassify an asthmatic patient comprising obtaining an expression profilefrom nasal respiratory epithelium of a human patient with asthma,comparing the patient's gene expression profile with a gene expressionprofile for stable asthma and a gene expression profile for exacerbatedasthma, and determining the patient's asthma classification based on atleast a two-fold difference between the compared expression profiles.35. The method of claim 34 further comprising providing prophylaxis ortreatment to the patient based on the classification.